Cytokinesis is a dramatic example of a cell shape change during which the mechanical constriction of the contractile ring leads to cell separation at the end of mitosis. The mechanical forces are largely generated by actin-based myosin-motor proteins. Cytokinesis is essential for normal cell proliferation and is of medical interest for its role in hyperproliferative diseases such as cancer. [unreadable] [unreadable] In Dictyostefium discoideum, genetic interactions have been identified between two actin bundling proteins that have complementary cellular distributions. Cortexillin I is localized to the contractile ring while dynacortin is cortically enriched but excluded from the contractile ring. Dynacortin is a novel dimeric actin bundling protein. A fragment of dynacortin (C181) disrupts the native dynacortin complex and suppresses the cytokinesis defect of cortexillin I mutant strains. The implication is that cells have evolved distinct actin cross-linking proteins with complementary cellular distributions that orchestrate cell shape changes. [unreadable] [unreadable] In this proposal, the mechanisms of actin binding/bundling by recombinantly expressed and purified full-length dynacortin, C181, and the heterodimer will be studied using a variety of equilibrium and kinetic techniques. Since dynacortin is phosphorylated in vivo, the protein will be purified from Dictyostefium and tested for its ability to bind and bundle actin filaments. Kinases that phosphorylate dynacortin will be purified to homogeneity by following their ability to phosphorylate dynacortin in vitro. These enzymes will be identified by mass spectroscopy and studied using gene knockout technology and fluorescence microscopy of GFP-fusion proteins. Purified enzymes will be used to phosphorylate recombinant dynacortin so that the effect of a particular phosphorylation state may be fully quantitated. Another protein, DdERM, which was also isolated in a suppressor screen of cortexillin I, will be studied. DdERM is related to the ezrin-radixinmoesin family of proteins, which tether the cortical actin cytoskeleton to the plasma membrane. We hypothesize that the DdERM will share this property. By studying this protein, the role of tethering of the actin cortical meshwork to the plasma membrane in cytokinesis may be evaluated. Finally, new genes involved in cortical shape control will be identified by saturating the genetic selection experiment for suppressors of cortexillin I and by screening for enhancers of myosin-II null cells.